Cloning, sequencing and expression of a gene encoding an eukaryotic amino acid racemase, and diagnostic, therapeutic, and vaccination applications of parasite and viral mitogens

ABSTRACT

A method of preventing or inhibiting infection by a parasite or virus in vivo comprising administering to a human in need thereof a parasite or virus mitogen in a sub-mitogenic amount sufficient to induce a protective immune response against the parasite or virus in the human.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. application Ser. No. 10/662,425, filedSep. 16, 2003, which is a continuation of U.S. application Ser. No.09/725,945, filed Nov. 30, 2000, and claims the benefit of U.S.Provisional Application Ser. No. 60/168,631, filed Dec. 3, 1999(attorney docket no. 03495.6044); U.S. Provisional Application Ser. No.60/220,207, filed Jul. 24, 2000 (attorney docket no. 3495.6054); andU.S. Provisional Application Ser. No. 60/221,117, filed Jul. 27, 2000(attorney docket no. 3495.6055), all of which are relied upon andincorporated by reference herein.

BACKGROUND OF THE INVENTION

This invention relates to the discovery of a new gene, which is thefirst isolated and cloned, that encodes an amino acid eukaryoticracemase. The invention covers, particularly, the Tc45 gene encoding aTrypanosoma cruzi-derived B-cell mitogen. The encoded protein also is aeukaryotic proline racemase. The invention also relates to a process ofproduction of D-amino acid using an eukaryotic amino acid racemase. Thisinvention also relates to the use of the protein encoded by the Tc45gene to induce a protective immune response against T. cruzi infectionin a human. This invention also relates to methods of using otherparasite mitogens and viral mitogens for inducing protective immunityagainst the corresponding parasitic or viral infections in humans.

The process of production of an D-amino acid by using a L-amino acidsource comprises the use of an eukaryotic amino acid racemase specificfor the amino acid of interest, the said racemase being produced from arecombinant expression system containing a vector having apolynucleotide sequence encoding the said enzyme. In prokaryotic hosts,the racemases are known to be implicated in the synthesis of D-aminoacids and/or in the metabolism of L-amino acids. Therefore, the presenceof free D-amino acids in tumors and in progressive autoimmune anddegenerative diseases suggests the biological importance of eukaryoticamino acid racemases. It is well known that proteins or peptidescontaining D-amino acids are resistant to proteolysis by host enzymes.In addition, such proteins containing D-amino acids, at least oneD-amino acid residue, can display antibiotic or immunogenic properties.

Isolation and characterization of molecules playing a key role inparasite metabolism, or in their interactions with the host immunedefense, are fundamental for the development of rational strategies forvaccination and therapy. Attempts to provide effective immunity toparasites are limited by poor specific immune responses to parasiteantigenic molecules in early phases of infection. Lymphocyte polyclonalactivation is a generalized mechanism of immune evasion amongstpathogens^(I). Such “parasite evasion” owes, at least in part, to therelease of mitogenic or superantigenic moieties that inhibit hostspecific responses by triggering polyclonal, parasite non-specificlymphocyte activation. The resulting non-specific immune responses areassociated with immunosuppression and autoimmunity, as observed in humanand experimental infections by the protozoan parasite Trypanosoma cruzi,the etiological agent of Chagas disease²⁻⁶.

To date, there is no effective treatment or vaccine against Trypanosomacruzi infection and Chagas disease pathology. Attempts to isolateimmunodominant protective epitopes have failed¹. Using a mouse model ofT. cruzi infection it has previously been shown that reduced levels ofpolyclonal lymphocyte responses correlate with resistance to infectionand cardiopathy^(2,7-9). As we have suggested, and has been demonstratedby Arala-Chaves^(3I) for Candida albicans infections, mitogenic moietiescan be used as vaccination targets to induce specific neutralization ofthe mitogen, thus aborting the microorganism “strategy” to deviateimmune responses into non-specific polyclonal activation andimmunosuppression. Understanding the mechanisms underlying“non-specific” lymphocyte activation may open the way for theirneutralization, and thus allow for effective immune responses againstinfectious agents. There is a need in the art for a molecule that couldbe an appropriate target for such attempts.

Furthermore, there is a growing interest in the biological role ofD-amino acids, either as free molecules or within polypeptide chains inhuman brain, tumors, anti-microbial and neuropeptides, as well as in“protein fatigue”³², suggesting widespread biological implications.Research on D-amino acids in living organisms has been hampered by theirdifficult detection. However, recent purification of a serine racemasefrom mammalian brain³³ indicates conservation throughout evolution.There also exists a need in the art for racemases that are specific forknown compounds.

SUMMARY OF THE INVENTION

This invention aids in fulfilling these needs in the art. Moreparticularly, this invention relates to the characterization of aparasite molecule implicated in polyclonal responses that may serve as anovel target for vaccination and therapy. After identifying a proteinwith B-cell mitogenic properties in culture supernatants of infectiveparasite forms, the corresponding gene was cloned and its genomicorganization was characterized. The protein has been characterized as acofactor independent proline racemase, with strong homology to theproline racemase isolated from Clostridium sticklandii, thus providingthe first report on an eukaryotic amino acid racemase gene.

In particular, this invention provides a purified peptide comprising anamino acid sequence (SEQ ID NOS: 1, 2, 3 and 4) encoded by the Tc45gene. This invention also provides polypeptide fragments derived fromSEQ ID NOS: 7, 8, 9, 10 and 11 containing at least 10 amino acids.

This invention additionally provides purified polynucleotides comprisingthe nucleic acid sequences of the Tc45 gene (SEQ ID NOS: 7, 8, 9, 10 and11). This invention also provides nucleic acid fragments derived fromSEQ ID NOS: 7, 8, 9, 10 and 11 containing 15 to 40 nucleotides.

Additionally, the invention includes a purified polynucleotide thathybridizes specifically under conditions of moderate stringency with apolynucleotide of SEQ ID NOS: 7, 8, 9, and 10.

SEQ. ID 7 represents the full nucleotide sequence encoding theTrypanosoma cruzi proline racemase and N-terminal signal sequence andthe 5′ and 3′ flanking non-coding regions.

The SEQ ID 8 represents the full nucleotide sequence and itscorresponding polypeptide sequences [including the N terminal signalsequence and the 3′ non-coding flanking region] coding for a prolineracemase of T. cruzi.

The construct as disclosed in SEQ ID 8 deleted of the 3′ non-codingflanking region and inserted in the PET28 vector (NOVAGEN), transformedin E. coli DH5 α was deposited at the CNCM under the accession numberI-2344.

A derived construct of I-2344 deleted of nucleotide sequencecorresponding to the signal peptide coding sequences, which is describedin SEQ ID 3, is used for the production of a recombinant active prolineracemase in E. coli. The E. coli DH 5α containing the plasmid with aninsert of 239 base pairs deposited at CNCM under accession numberI-2221, was obtained after amplification of the region by PCR techniquewith the primers SEQ ID Nos. 12 and 13. The insert was cloned into pTOPO II commercialized by INVITROGEN and then transformed in E. coli.

The invention further includes polynucleotide fragments comprising atleast 10 nucleotides capable of hybridization under conditions ofmoderate stringency conditions with any one of the nucleotide sequencesenumerated above.

In another embodiment of the invention, a recombinant DNA sequencecomprising at least one nucleotide sequence enumerated above and underthe control of regulatory elements that regulate the expression ofracemase activity in a host is provided.

The invention also includes a recombinant host cell comprising apolynucleotide sequence enumerated above or the recombinant vectordefined above.

In still a further embodiment of the invention, a method of detectingparasitic strains that contain the polynucleotide sequences set forthabove is provided.

Additionally, the invention includes kits for the detection of thepresence of parasitic strains that contain the polynucleotide sequencesset forth above.

The invention also contemplates antibodies recognizing peptide fragmentsor polypeptides encoded by the polynucleotide sequences enumeratedabove.

Still further, the invention provides for a screening method for activemolecules for the treatment of infections due to parasites, particularlyT. Cruzi, based on the detection of activity of these molecules onparasites.

This invention further provides an immunizing composition containing atleast a purified protein, or a fragment thereof, capable of inducing animmune response in vivo. The immune response can be a mitogenicpolyclonal immunoresponse in vivo. The immunizing composition issuitable for use against a parasite infection under sub-mitogenic doses.

This invention also provides a process to access the mitogenicity of amolecule called mitogen and the procedures to determine thesub-mitogenic dose suitable as an immunizing composition for use againsta parasite infection.

A vaccine composition of the invention for use against a T. cruziinfection comprises a purified 38 to P45 kda protein or a fragmentthereof.

A method of inhibiting an eukaryotic protein with an amino acid racemaseactivity according to the invention comprises treating a patient byadministering an effective amount of a molecule that inhibits theeukaryotic protein. The parasite can be T. cruzi.

This invention also provides a process for screening a molecule capableof inhibiting the amino acid racemase activity of an eukaryotic proteincomprising the steps of:

-   -   contacting the purified eukaryotic racemase protein with        standard doses of a molecule to be tested;    -   measuring inhibition of racemase activity; and    -   selecting the molecule.

BRIEF DESCRIPTION OF THE DRAWINGS

This invention will be more fully described with reference to thedrawings in which:

FIG. 1 depicts the proliferative activity of total spleen cellsstimulated in vitro by a 45 kDa B-cell polyclonal activator isolatedfrom parasite culture supernatants.

FIG. 1A depicts the proliferative activity of total spleen cellsstimulated in vitro by increasing concentrations (below graph) of totalproteins from culture supernatants of metacyclic trypomastigotesdifferentiated in vitro. The inset shows proliferation of total (▪) or Tcell-depleted (/) splenocytes in the presence of proteins from totalculture supernatant c.p.m., counts per minute.

FIG. 1B depicts proliferative activity of total spleen cells stimulatedin vitro by HPLC pooled fractions at 24 h (▪), 48 h (□) and 72 h (/).³H-thymidine uptake (c.p.m., counts per minute) after 48 h in thepresence of concavalin A and lipopolysaccharide was 32,000 and 3,500counts per minute, respectively. To rule out the possibility ofmitogenic effect due to lipopolysaccharide contamination in the samples,proliferation assays were also done using freshly recovered splenocytesfrom the ‘lipopolysaccharide non-responder’ mouse strain C3H/He; thesame levels of proliferation were obtained.

FIG. 1C shows an 8% SDS-PAGE analysis of fractions 21-24 (silverstaining). Molecular size markers are shown at the left margin. Thearrow corresponds to a 45 kDa band.

FIG. 2 shows the homology or similarity between the Tc45 protein (Tc)[SEQ ID NO: 1] and the bacterial proline racemases Clostridiumsticklandii (Cs) [SEQ ID NO.:5] and Pseudomonas aeruginosa (Pa) [SEQ IDNO.:6]. The computer-predicted signal peptide is indicated by a doublearrow; peptide sequences obtained by microsequencing appear underlined;peptides used for designing degenerate primers appear in italiccharacters. Proline racemase active sites are boxed; dashes indicategaps generated for best fit.

FIG. 3 shows the genomic organization and transcription of the Tc45gene.

FIG. 3A shows a Southern blot analysis of 5 μg of T. cruzi genomic DNAdigested with indicated restriction enzymes and hybridized with a³²P-labeled probe, including the TcPA45 coding sequence, is shown.Molecular weights are indicated.

FIG. 3B is a Northern blot analysis of 20 μg of total RNA fromepimastigotes forms hybridized with a ³²P-labeled probe as above.Molecular weights [Kb] are indicated.

FIG. 3C shows mRNA expression of TcPA4S in different life stages of theparasite, shown by electrophoresis of gene fragments obtained byspecific reverse transcription from total parasite RNA followed by PCRamplification using the sequences of the mini-exon (spliced leader) andR-300-45 primers and subsequent amplification of a 170-bp internalfragment. M1, M2 and M3, molecular size markers (sizes, left and rightmargins) are shown. First-strand cDNA reactions were done in thepresence (+) or absence (−) of reverse transcriptase, to exclude thepossibility of further PCR amplification of fragments due to genomic DNAcontamination, C1 and C2, internal negative (no template) controls; E,epimastigote, T, typomastigote and M, metacyllic.

FIG. 4 shows the results of characterization of rTcPA45 activities invitro.

FIG. 4A shows an 8% SDS-PAGE gel of rTcPA45 (Coomasie blue staining).

FIG. 4B depicts proliferative activity of total splenocytes (5×10⁴cells/well) in the presence of increasing concentrations of rTcPA45(μg/ml).

FIG. 4C depicts percent racemisation of L-proline, D-proline,L-hydroxy-proline, D-hydroxy-proline substrates. Reaction conditions are0.2 M Na-acetate/25 mM β-mercaptoethanol buffer, pH6, rTcPA45 (3 μg/ml),30 min incubation at 37° C. in 500 μl. The reaction was stopped byincubating for 10 min at 80° C.

FIG. 4D depicts percent inhibition of racemization of 80 mM L-proline inthe presence of several inhibitors.

FIG. 4E depicts percent racemization of L-proline (80 mM) as a functionof pH (buffers used were 0.2 M: Na-acetate, K-phosphate and Tris-HCl andcontained 25 mM β-mercaptoethanol).

FIG. 5 shows the nucleotide sequence [SEQ ID NO: 17] and peptidesequence [SEQ ID NO: 1] of TcPA45. The polypyrimidine rich region,splice leader acceptor sites, signal peptide, and polyadenylation siteare indicated. The peptide sequences obtained by microsequencing of thenative TcPA45 protein are underlined.

FIG. 6 is a demonstration of a cytosolic proline racemase inepimastigote forms of T. cruzi.

-   Western blot: membranes containing in:-   Lane 1: total epimastigote extract from 5×10⁵ epimastigote forms-   Lane 2: Epimastigote culture supernatant-   Lane 3: Soluble fraction of epimastigote extract (cytosolic) from    5×10⁵ epimastigote forms.-   Lane 4: Insoluble fraction of epimastigote extract from 5×10⁵    epimastigote forms-   Lane 5: as in lane 1, from 10× more parasite forms (5×10⁶    epimastigotes)-   Lane 6: as in lane 3, from 10× more parasite forms (5×10⁶    epimastigotes)-   Lane 7: as in lane 2-   Lane 8: as in lane 4, from 10× more parasite forms (5×10⁶    epimastigotes) Membranes were incubated with mouse polyclonal    antibodies raised against the rTcPA45 protein (primary antibody).    Second step reaction was done with goat anti-mouse IgG-horseradish    peroxidase (human absorbed). Reactivities were developed by    ECL-chemiluminescence Amersham kit. FIG. 6 represents 10 seconds    exposure of the film.

FIG. 7 depicts the results of an ELISPOT assay. Spots correspond toimmunoglobulin-producing B-cell (of a particular isotype) directed tothe coated antigen (here: goat anti-mouse immunoglobulins or rTcPA45).See also Example 10 and Table 1.

FIGS. 8A, 8B, 8C, 8D, and 8E depict the results of DNA vaccination usingvarious DNA constructs. See also Example 11.

FIG. 9 depicts the characterization of rTcPA45 mitogenic activity.Proliferative activity of total splenocytes obtained from athymic oreuthymic mice in the presence of rTcPA45 or lipopolysaccharide is shownat 24 h (□), 48 h (/) or 72 h (▪).

FIG. 10 depicts the differential expression of rTcPA45 protein in theparasite.

FIG. 10A depicts the cellular localization of the Tc45 protein indifferent life stages of the parasite, shown by indirectimmunofluorescence using polyclonal mouse serum against rTcPA45 followedby staining with the Alexa 488™ goat antibody against mouse IgG (H+ L),F(ab′)₂ fragment conjugate (bottom row), compared with control stainingusing the Alexa 488 ™ F(ab′)₂ fragment conjugate alone (top left) orafter incubation of the parasites with serum from chronically infectedmice (Chronic serum).

FIGS. 10B and 10C depict the detection of Tc45 protein in total extractsof epimastigote (E), metacyclic (M) and trypomastigote (T) forms of theparasite, compared with recombinant rTcPA45 (R) protein, by western blotanalysis. Arrows, calculated molecular weights for isoforms of theTcPA45 (R) protein.

FIG. 10D depicts the presence of the 39-kDa isoform of the Tc45 proteinin total (Et), soluble (Ese) and insoluble (Emb) sonic extracts ofnon-infective epimastigote forms of the parasite, compared with itsabsence in culture supernatants (Ecs). Molecular sizes (b-d) are shownat the left margins.

FIG. 11 depicts the correlation between mitogenic and racemaseactivities.

FIG. 11A depicts the proliferative activity of total mouse splenocytesin the presence of rTcPA45 protein that is enzymatically active(rTcPA45) or lacking racemase activity by being heated (80°-rTcPA45), bylong term storage at 4° C. (Ina-rTcPA45), or by pre-incubation ofrTcPA45 with pyrrole-Z carboxylic acid (rTcPA45+1 mM PCA), Iodoacetamide(rTcPA45+1 mM IAA) or iodoacetate (rTcPA45+1 mM IAC) inhibitors,compared with 1 mM pyrrole-2-carboxylic acid 1 mMPCA) alone.

FIG. 11B depicts the competitive inhibition of rTcPA45-inducedproliferative activity of total mouse lymphocytes by increasingconcentrations of L- or D-proline substrates. Shown are controls,cultures of splenocytes with 50 mM L- or D-proline alone. c.p.m., countsper minute.

FIG. 12 depicts the results of Western blot of mouse specific immuneresponses directed to mitogenic rTcPA45 following immunization. See alsoExample 15.

FIG. 13 depicts two dose-response curves for two different hypotheticalmitogens, A and B, in an assay of proliferative activity describedhereinafter.

FIG. 14 depicts the results of immunization according to the invention.

FIG. 14 A. DNA vaccination. 8 week old BALB/c mice (5 mice/group) wereinjected 3 times (i.m., 50 μg of plasmid/femoral quadriceps) at 3 weeksinterval with the following constructs: pcDNA3 vector alone as control,or pcDNA3 containing the full encoding sequence of the TcPA45 gene with(Long) or without (Short) the fragment encoding the signal peptide. Micewere challenged 4 weeks after the last injection with 10⁴ infectiveforms of the parasite/mouse, and the parasitemia was scored during 35days.

OBS. It is worth noting that BALB/c mice were treated by almost 2 months(9 weeks) to follow the vaccination protocol and were challenged at 21weeks of age. Its is well known that mice of more than 9 weeks of ageare naturally more resistant to the experimental infection withTrypanosoma cruzi and no morality is observed.

The results using this vaccination protocol revealed that 3 injectionsof pcDNA3 containing either the Short or Long encoding sequenvces of theTcPA45 gene, are able to reduce by more than 85% the parasitemia levels.

FIG. 14B. rTcPA45 injection. 6 week old BALB/c mice were injectedintraperitoneally (i.p.) with 10 ηg of rTcPA45 and then boosted i.p. oneweek later with 50 μg of rTcPA45. Mice were challenged 1 week after theboost with 10⁴ infective forms of the parasite/mouse, and theparasitemia was scored during 30 days. Control mice did not receive anytreatment before infection.

FIG. 15 depicts SEQ ID NO: 2, an amino acid sequence encoded by the Tc45gene with markings as described for FIG. 2.

FIG. 16 depicts SEQ ID NO: 4, an amino acid sequence encoded by the Tc45gene with markings as described for FIG. 2.

FIGS. 17A and 17B depict SEQ ID NO: 7, a nucleic acid sequence of Tc45with markings as described for FIG. 5.

FIGS. 18A and 18B depict SEQ ID NO: 8, a nucleic acid sequence of theTc45 gene with markings as described for FIG. 5.

FIGS. 19A and 19B depict SEQ ID NO: 9, a nucleic acid sequence of theTc45 gene with markings as indicated for FIG. 5.

FIG. 20 depicts SEQ ID NO: 10, a nucleic acid sequence of the Tc45 genewith markings as indicated for FIG. 5.

FIGS. 21A and 21B depict SEQ ID NO: 11, a nucleic acid sequence of theTc45 gene with markings as indicated for FIG. 5.

DETAILED DESCRIPTION

Having noted that proteins released from trypomastigote forms ofTrypanosoma cruzi behave as polyclonal B-cell activators¹⁰, it washypothesized that infective metacyclic forms would display an increasedproduction of mitogenic molecules, thereby promoting such a mechanism ofimmune evasion. To investigate the release of proteins with B-cellmitogenic activity by T. cruzi, culture supernatants of in vitrodifferentiated metacyclic trypornastigotes in a protein-free definedmedium were produced¹¹. Lymphocyte mitogenic activity in totalconcentrated culture supernatants was confirmed by lymphocyteproliferation assays.

Briefly, freshly recovered splenocytes from 8 week old male Balb/c miceseeded at a concentration of 5×10⁴ cells/well were incubated for 24, 48and 72 h in 5% FCS RPMI-1640 medium. A 16 h ³H-thymidine pulse at 1μCi/well was performed before harvesting. ³H-Thymidine uptake wasdetermined in a beta-plate liquid scintillation counter (LKB-Wallac).All points were done in triplicate and the corresponding standarddeviation calculated. For comparison, ³H-thymidine uptake after 48 h inthe presence of ConA (10 μg/ml) and LPS (5 μg/ml) was 32000 c.p.m. and3500 c.p.m., respectively. T-cell depletion was performed by incubatingfreshly recovered spleen cells for 30 min at 37° C. in the presence ofanti-Thy 1.2 monoclonal antibody (Cedarlane) and rabbit complement(Cedarlane). In order to rule out a possible mitogenic effect due to LPScontamination in samples, proliferation assays were also performed onfreshly recovered splenocytes from the LPS non-responder mouse strainC3H/Hej. Same levels of proliferation were verified. Balb/c mice werepurchased from Charles River, France. C3H/Hej mice were maintained inour animal facilities.

Proliferation is sustained over a 72 h period of culture in a dosedependent manner as shown in FIG. 1 a. Increasing concentrations (μgprotein/ml) of total culture supernatant of in vitro differentiatedmetacyclic trypomastigotes are shown in this Fig. The inset showsproliferation of total or T-cell depleted splenocytes in the presence of0.6 μg/ml of total culture supernatant. As for other B-cell mitogens,the same level of proliferation was observed when supernatants weretested on T-cell depleted splenocytes (FIG. 1 a).

To identify the molecules responsible for mitogenic activity, theparasite proteins present in the metacyclic culture supernatants werefractionated by HPLC anion-exchanger chromatography, and the resultingfractions were tested as above. Fractions 22-24 (eluted at 368mM<NH₄-acetate<467 mM) showed the highest ³H-thymidine uptake at 24hours. The results are shown in FIG. 1 b. 0.5 μg/ml of HPLC pooledfractions were resuspended in RPMI.

Previous observations using DEAE-chromatography-purified parasiteculture supernatants had shown that a protein fraction of 40-45 kDa wasable to induce B-cell activation and proliferation in vivo. Theestimation of this range of molecular weight was done more or less 10%and compared with standard molecular weight kit markers commercializedby BioLabs (USA).

SDS-PAGE analysis of the HPLC fractions revealed the presence of aprotein with an apparent molecular weight around 45 kDa only infractions 21-24 as shown in FIG. 1 c. (8% SDS-PAGE gel of fractions 21to 24 (silver staining)). The isoelectric point of the protein wasestimated between 4.5 and 5.0 by isoelectric focusing. These dataindicated that a protein with B-cell mitogenic activity was present infractions 21-24 eluted from the anionic matrix, and the 45 kDa proteinband was thus selected as the main candidate.

To obtain peptide sequences from the 45 kDa protein, which wasidentified as Tc45, and allow for subsequent PCR-assisted cloning of agene fragment, the 45 kDa protein band was isolated by SDS-PAGE, and itsinternal digestion and microsequencing were undertaken. Six peptideswere isolated, sequenced, and were shown to have the followingsequences: (W)⁴³II⁴⁶ [SEQ ID NO:18]; I⁹⁰VTGSLP(D)I(S)G¹⁰⁰ [SEQ IDNO:19]; A ¹⁸³TNVPVVLDTPAGLVR¹⁹⁸ [SEQ ID NO:20]; V²⁴¹DIAFGGNF²⁴⁹ [SEQ IDNO:21]; N³¹⁶VVIFGNR³²³ [SEQ ID NO:22], and M³³⁸ATLYAK³⁴⁴ [SEQ ID NO:23].These sequences are underlined in FIG. 2.

Peptides 4 [SEQ ID NO:21] and 5 [SEQ ID NO:22] were selected fordegenerate primer design on the basis of the relatively low level ofdegeneracy in their corresponding coding sequences. The sequences ofthese peptides are identified by italic characters in FIG. 2.

Reverse transcription was performed on total RNA from T. cruzitrypomastigote forms using reverse degenerate primers for both peptides.The resulting cDNA was then used as a template for PCR amplification.From all possible combinations, when using in the PCR reaction forwardprimer for peptide 4 (SEQ ID No. 13), reverse primer for peptide 5, andtemplate cDNA synthesized with reverse primer for peptide 5 (SEQ ID No.12), only one PCR product of 239 bp was shown to contain both primersafter cloning and sequencing. The sequence analysis revealed that thefragment contained an unique open reading frame (ORF) flanked bypeptides 4 [SEQ ID NO:21] and 5 [SEQ ID NO:22] coded in frame.

To obtain the full sequence of the Tc45 gene, the ³²P-labelled 239 bpPCR product was used as a probe to screen a Trypanosoma cruzi cloneCL-Brener lambda Fix II genomic library. Four independent positivephages were isolated. Restriction analysis and Southern blothybridization revealed two types of patterns, each represented by twophages, suggesting that the Tc45 gene is present in at least two copiesper haploid genome.

The complete sequence of the Tc45 gene and flanking regions (GenBankaccession no. AF195522), revealed an ORF of 423 codons containing allsequenced peptides (FIG. 2) [SEQ ID NO:1]. Computer analysis predicts a29 amino acid signal peptide [SEQ ID NO: 3] (double arrowed in FIG. 2)suggesting active secretion by T. cruzi. This is in agreement with thefact that the protein was purified from culture supernatants. Apoly-pyrimidine rich region and probable trans-splice acceptor site isobserved 56 and 7 base pairs (bp) upstream of the ATG codon,respectively. Interestingly, an alternative trans-splicing signal ispresent about 170 bp upstream of the second ATG codon within the codingregion, which if used would allow the expression of a truncated,non-secreted protein lacking 69 amino acids, which is SEQ ID No. 4.Polyadenylation may take place at positions 1442 or 1443, which arepreceded by repeats of the triplet UUA, a motif found at the 3′ end ofother T. cruzi genes^(I2,13).

To investigate the genomic organization and transcription of the Tc45gene, Southern and Northern blot analyses were done. FIG. 4 depicts theresults of characterization of rTcPA45 activities. FIG. 4, part a, is an8% SDS-PAGE gel of rTcPA45 (Coomasie blue staining). Part b depicts theproliferative activity of total splenocytes (5×10⁴ cells/well) in thepresence of increasing concentrations of rTcPA45 (μg/ml). Part c showspercent racemization of L-proline, D-proline, L-hydroxy-proline,D-hydroxy-proline substrates. Reaction conditions were: 0.2 MNa-acetate/25 mM β-mercaptoethanol buffer, pH6, rTcPA45 (3 μg/ml), 30min incubation at 37° C. in 500 μl. The reaction was stopped byincubating for 10 min at 80° C. FIG. 4, part d, shows percent inhibitionof racemization of 80 mM L-proline in the presence of severalinhibitors. Part e shows percent racemization of L-proline (80 mM) as afunction of pH (buffers used were 0.2 M: Na-acetate, K-phosphate andTris-HCl and contained 25 mM β-mercaptoethanol). Reactions were carriedout for 30 min at 37° C. and stopped for 5 min at 80° C. All reagentsand inhibitors were purchased from Sigma. The shift in optical rotationwas measured in a Polarimeter 241 MC (Perkin Elmer).

To investigate the genomic organization and transcription of the Tc45gene, we used Southern blot analysis; this indicated the presence of twogene copies per haploid genome. There are probably two homologous Tc45genes (FIG. 3 a). Digestion with BamHI and BglII producted twohybridizing bands, consistent with the presence of two gene copies, asthe probe has neither enzyme restriction site. High-molecular-weight DNAhybridized after probable partial digestion with SalI, consistent withthe absence of this site within the coding sequence covered by theprobe. Both PstI and TaqI cleaved within the probe and produced morethan one hybridizing band per gene copy. Preliminary results indicatedthat they are located on different chromosomes (data not shown).Northern blot analysis on total RNA from epimastigotes showed atranscript of around 1.5 kb, as expected from the genomic sequence (FIG.3 b). We confirmed the presence of the Tc45 mRNA in different parasiteforms by reverse transcription PCR using primers specific for the Tc45gene (FIG. 3 c). Several point mutations were already identified in thesequence of the putative Tc45-B gene copy representative of the secondphage type (FIG. 2), and further transcriptional and functional analysesof the alleles are underway.

To identify homologies with other genes, we compared the Tc45-A genecopy and protein sequence to several databases. There was homology(nucleotide sequence, 57.7%; IDENTITY amino-acid sequence, 52.4%IDENTITY) with the only proline racemase described¹⁴, an intracellularhomodimetric protein isolated from Clostridum sticklandii. This enzymecatalyzes the interconversion between the L- and D-proline enantiomers,and its reaction mechanism has been studied extensively¹⁴. There wasalso homology with the translation of an ORF sequence from Pseudomonasaeruginosa in contig 53 of the unfinished Pseudomonas genome project(amino-acid sequence, 37.9% IDENTITY; C. sticklandii proline racemaseand the translation of the P. aeruginosa ORF, 47% IDENTITY. The C.sticklandii proline racemase active site has been identified¹⁵ and isconserved in the Tc45 protein. Homology with both bacterial proteinsstarted at amino acid 70 of Tc45 (FIG. 2). The presence of the KIIKpeptide (FIG. 2, underlining) in the Tc45 protein purified from parasiteculture supernatants confirmed the presence of the extra N-terminalportion of the protein released by T. cruzi metacyclic forms.

More particularly, the SDS-PAGE analysis of the over-expressed andpurified protein is shown in FIG. 4 a. Using in vitro proliferationassays of naive murine spleen cells, recombinant protein rTc45 was shownto display a similar mitogenic activity to the one observed with thenative protein fraction, purified from culture supernatants. Thus,rTcPA45 (for T. cruzi polyclonal activator 45) induces spleen lymphocyteproliferation, which increases with time over a 72 h period of culture(FIG. 4 b). Proliferation is dose dependent, with a bell-shaped responsecurve (starting from 0.8 μg/ml, and peaking at 50 μg/ml) typical of allmitogens described to date. rTcPA45 is indeed a T cell-independentpolyclonal activator of B lymphocytes, as shown by the magnitude andincrease of the proliferative response of total spleen lymphocytesobtained from athymic mice compared with the response of lymphocytesfrom euthymic individuals (FIG. 9). Injection of 50 μg rTcPA45 in vivoinduced a 2 fold increase in spleen cell numbers by day 4, accompaniedby an increase in numbers of immunoglobulin (Ig)-secreting B cells ofthe IgM, IgG2a, IgG2b and IgG3 isotypes (2.5 fold to 100×), whileshowing a complete lack of rTcPA45-specific Ig-secreting B cells,indicating the polyclonal B-cell mitogenicity of the protein (data notshown).

Injection of 50 μg of rTcPA45 in vivo induces a 2-fold increase inspleen cell numbers by day 7 accompanied by 2.5 to 4 fold increase innumbers of Ig-secreting B cells of IgM, IgG2a, and IgG2b isotypes, whileshowing a complete lack of Ig-secreting B cells directed to TcPA45demonstrating the polyclonal B-cell mitogenicity of the protein as shownin Example 10, Table I.

To confirm that the TcPA45 protein is indeed a proline racemase, invitro biochemical assays were performed to measure the shift in opticalrotation of either L- or D-proline substrates. As can be seen in FIG. 4c, rTcPA45 racemises both L- and D-proline, but not L- orD-hydroxy-proline, nor any other natural L-amino acids. Such rTcPA45racemase activity is co-factor independent, notably of pyridoxalphosphate, and thus closely resembles the C. sticklandii prolineracemase^(I4).

Furthermore, the rTcPA45 enzymatic activity is inhibited to differentextents by the presence of previously described inhibitors¹⁴, such asmaleic acid, iodoacetamide, iodoacetate, and pyrrole-2-carboxylic acid(FIG. 4 d). Interestingly, rTcPA45 proline racemase activity is maximalat pH 6 (FIG. 4 e), two units lower than that of the bacterialenzyme^(I4). The optimal temperature for enzymatic activity is 37° C.,and the enzyme is inactivated by 10 min heating at 80° C. (data notshown).

To analyze the cellular localization of the parasite TcPA45, we usedimmunofluorescence experiments with a polycyclonal serum raised againstrTcPA45. Whereas serum from chronically infected mice stainedtypomastigote cells uniformly, rTCPA45 specific antibodies stainedmostly the cytoplasm of epimastigote forms but not the nucleus or thekinetoplast (FIG. 10 a). In vitro-differentiated metacyclic forms showeda less in tense and more diffuse pattern of cytoplasmic staining thandid epimastigotes. However, bloodstream trypomastigote forms werestrongly labeled at the flageliar pocket and the anterior and posteriorends of the parasite, and lightly along the flagellum and cytoplasm.These experiments substantiate the hypothesis that T. cruzi has anintracellular form of the proline racemase and the secretion might onlytake place in the infective forms. Western blot analysis of cellextracts of the parasite confirmed that the TcPA45 protein was presentin different developmental stages (FIG. 10 b). We detected a TcPA45protein around 39 kDa in molecular mass in epimastigotes (non-infectiveinsect forms) and 41.S kDa in infective metacyclic trypomastigotes,compared with the computer-predicated molecular masses of 43.4 kDa and38 kDa, respectively, for a secreted and a non-secreted form of theprotein (FIG. 10 c). Western blot analysis of the non-infectiveepimastigote cell stage showed Tc45 proline racemase mostly in thesoluble cellular fraction, only weakly in the cellular insolublefraction and absent from culture medium (FIG. 10 d).

To confirm the relationship, if any, between the enzymatic and themitogenic activities of rTcPA45, we used in vitro proliferation assayswith active rTcPA45 and different forms of the inactivated enzyme.Unexpectedly, mitogenic activity was abolished when rTcPA45 wasinactivated by being heated or by long storage at 4° C., or wheneverenzymatic inhibition was achieved by pre-incubation of the protein withspecific (pyrrole-2-carboxylic acid) or nonspecific (iodoacetamide andiodoacetate) inhibitors of proline racemase (FIG. 11 a). Mitogenicactivity was also affected considerably by supplementation of thecultures with increasing amounts of L- or D-proline, in a dose-dependentmanner, indicating that competitive inhibition occurred in the presenceof specific substrates (FIG. 11 b). There was no cell proliferation whenlymphocytes were cultured in the presence of 50 mM L- or D-proline alone(FIG. 11 b). Furthermore, mitogenic activity due to another B-cellmitogen was unaffected by the inhibitors or substances (data not shown).Although the antibodies against TcPA45 raised against the recombinantprotein were not able to inhibit racemization or to neutralize mitogenicactivity in vitro, and thus cannot be used to support a link betweenthese activities, the results indicate that a free and intact activesite of the rTcPA45 protein is necessary to allow mitogenicity.

This is the first description of an amino acid racemase gene in aneukaryotic organism. Thus, this invention relates to the biochemicalisolation, cloning, and molecular characterization of a B-cell mitogenreleased by Trypanosoma cruzi. Unexpectedly, this is also the firstdescription of a racemase in a parasite.

In bacteria, amino acid racemases are cytoplasmic proteins participatingin metabolic processes or in the synthesis of post-translationallymodified peptides¹⁶. It is known that T. cruzi can use L-proline as amajor carbon source¹⁷, possibly through a D-proline intermediate¹⁸. Inthis case, however, one might expect to find a cytosolic prolineracemase. Indeed, we have identified a cytosolic proline racemase inepimastigote forms of T. cruzi as shown in FIGS. 6 and 10(b). Thisracemase presents a molecular weight of 38 kd in 10% SDS Page by using astandard molecular weight kit commercialized by BioLABS (USA). Theestimation of the molecular weight is done more or less 10% around 38kda. It is well established that proteins bearing D-amino acids arehighly resistant to eukaryotic proteases. Thus, it might also bepossible that the parasite uses the racemization mechanism duringmetacyclogenesis to synthesize and express, on its surface, proteinscontaining D-proline, therefore ensuring a certain degree of resistanceto host-induced proteolytic mechanisms during cell invasion.Interestingly, T. cruzi differentiation from epimastigote totrypomastigote is induced in the presence of L-proline at pH 6 in theinsect's gut¹⁹ as well as during in vitro metacyclogenesis¹¹, and theproline racemase activity of the TcPA45 protein might be involved inthis process.

FIG. 2 shows homology between the Tc45 protein (Tc) and the bacterialproline racemases Clostridium sticklandii (Cs) and Pseudomonasaeruginosa (Pa). Identical residues appear in bold characters; thecomputer-predicted signal peptide is indicated by a double arrow;peptide sequences obtained by microsequencing appear underlined;peptides used for designing degenerate primers appear in italiccharacters. Proline racemase active sites are boxed; dashes indicategaps generated for best fit.

The homology of TcPA45 protein sequence with the Clostridium sticklandiiproline racemase starts at amino acid 70 (second methionine in the ORFof TcPA45 gene) allowing the speculation that T. cruzi has acquired anextra 70 amino acid sequence N-terminal to the ancestral prolineracemase (comprising a signal peptide for secretion) gaining the abilityto produce either a cytosolic or a secreted form of the protein bydifferential trans-splicing of the mRNA. In addition, this process mightbe differentially regulated at distinct developmental stages of theparasite. In this context, it could then be hypothesised thatepimastigote forms produce a cytosolic protein, while infectivetrypomastigotes secrete the same protein that activates B-cellpolyclonal responses.

Interestingly, the disruption of alanine racemase and the D-amino acidaminotransferase genes of Listeria monocytogenes results in theinability of the bacteria to grow within the eukaryotic host cells²⁴.Both these gene products are involved in the synthesis of D-alanine thatis required for the production of a mucopeptide component of the cellwalls of virtually all bacteria. It remains to be investigated whetheror not such bacterial molecules are B-cell mitogens. Accordingly,working with random amino acid polymers, Sela and co-workers haveestablished that multichain polypeptides composed of D-amino acidsinduce antibody responses in a T-cell independent manner^(25,26), aproperty that can be interpreted as equivalent to B-cellmitogenicity^(27,28). Resistance of such D-amino acid peptides todegradation by host enzymes²⁹ could also explain the persistence ofpolyclonal responses (and immunosuppression)³⁰, even if parasiteproduction of the mitogen would be transient at the start of infection.

Thus, novel polynucleotides corresponding to the Tc45 gene from CLstrain (representative of lineage T. cruzi II). T. Cruzi have beenisolated and sequenced. The presence of the Tc45 gene has also beendemonstrated in a representative strain of another major lineage of T.cruzi [strain DM 28c, lineage T. cruzi I]. For a review onrecommendations on T. cruzi strain nomenclature see: Mem. Institut. OSW.Cruz, Vol. 94, Suppl. 1, page 429-432, 1999. These polynucleotidesinclude SEQ ID NOS: 7, 8, 9, 10, and 11. By “polynucleotides” accordingto the invention is meant the sequences referred to as SEQ ID NOS: 7, 8,9, 10, and 11, and the complementary sequences and/or the sequences ofpolynucleotides that hybridize to the referred sequences underconditions of moderate stringency. The moderate stringency conditionsare defined as washing conditions in 2×SSC at 55° C., and hybridizationoperated in 5×SSC at 55° C.

By “active molecule” according to the invention is meant a moleculecapable of inhibiting the activity of the purified recombinant or nativepolypeptide as defined in the present invention.

Thus, the polynucleotides of SEQ ID NO: 7 and its fragments can be usedas probes or to select nucleotide primers notably for an amplificationreaction, such as the amplification reactions further described. PCR isdescribed in the U.S. Pat. No. 4,683,202 granted to Cetus Corp. Theamplified fragments may be identified by agarose or polyacrylamide gelelectrophoresis, or by a capillary electrophoresis, or alternatively bya chromatography technique (gel filtration, hydrophobic chromatography,or ion exchange chromatography). The specificity of the amplificationcan be ensured by a molecular hybridization using as nucleic acid probesthe polynucleotides derived from SEQ ID NO: 7 and its fragments,oligonucleotides that are complementary to these polynucleotides orfragments thereof, or their amplification products themselves.

Amplified nucleotide fragments are useful as probes in hybridizationreactions in order to detect the presence of one polynucleotideaccording to the present invention or in order to detect the presence ofa parasite of T. Cruzi strain carrying genes encoding racemase activity,in a biological sample. This invention also provides the amplifiednucleic acid fragments (“amplicons”) defined herein above. These probesand amplicons can be radioactively or non-radioactively labeled, usingfor example enzymes or fluorescent compounds.

Preferred nucleic acid fragments that can serve as primers according tothe present invention are the following: 5′TTICCRAADATIACIACGTT3′ [SEQID NO: 12] 5′ATHGCITTYGGIGGIAAYTTT3′ [SEQ ID NO: 13]5′TTICCRAADATIACIACGTT3′ [SEQ ID NO: 14]5′CTCTCCCATGGGGCAGGAAAAGCTTCTG3′ [SEQ ID NO: 15]5′CTGAGCTCGACCAGATCTATCTGC3′. [SEQ ID NO: 16]The primers can also be used as oligonucleotide probes to specificallydetect a polynucleotide according to the invention.

Other techniques related to nucleic acid amplification can also be usedalternatively to the PCR technique. The Strand DisplacementAmplification (SDA) technique (Walker et al., 1992) is an isothermalamplification technique based on the ability of a restriction enzyme tocleave one of the strands at a recognition site (which is under ahemiphosphorothioate form), and on the property of a DNA polymerase toinitiate the synthesis of a new strand from the 3N OH end generated bythe restriction enzyme, and on the property of this DNA polymerase todisplace the previously synthesized strand being localized downstream.

The SDA amplification technique is more easily performed than PCR (asingle thermostated water bath device is necessary), and is faster thanthe other amplification methods. Thus, the present invention alsocomprises using the nucleic acid fragments according to the invention(primers) in a method of DNA or RNA amplification, such as the SDAtechnique. The polynucleotides of SEQ ID NO: 7 and its fragments,especially the primers according to the invention, are useful astechnical means for performing different target nucleic acidamplification methods, such as:

-   -   TAS (Transcription-based Amplification System), described by        Kwoh et al. in 1989;    -   SR (Self-Sustained Sequence Replication), described by Guatelli        et al. in 1990;    -   NASBA (Nucleic acid Sequence Based Amplification), described by        Kievitis et al. in 1991; and    -   TMA (Transcription Mediated Amplification).

The polynucleotides of SEQ ID NO: 7 and its fragments, especially theprimers according to the invention, are also useful as technical meansfor performing methods for amplification or modification of a nucleicacid used as a probe, such as:

-   -   LCR (Ligase Chain Reaction), described by Landegren et al. in        1988 and improved by Barany et al. in 1991, who employ a        thermostable ligase;    -   RCR (Repair Chain Reaction), described by Segev et al. in 1992;    -   CPR (Cycling Probe Reaction), described by Duck et al. in 1990;        and    -   Q-beta replicase reaction, described by Miele et al. in 1983 and        improved by Chu et al. in 1986, Lizardi et al. in 1988, and by        Burg et al. and Stone et al. in 1996.

When the target polynucleotide to be detected is RNA, for example mRNA,a reverse transcriptase enzyme can be used before the amplificationreaction in order to obtain a cDNA from the RNA contained in thebiological sample. The generated cDNA can be subsequently used as thenucleic acid target for the primers or the probes used in anamplification process or a detection process according to the presentinvention.

Nucleic acid probes according to the present invention are specific todetect a polynucleotide of the invention. By “specific probes” accordingto the invention is meant any oligonucleotide that hybridizes with thepolynucleotide of SEQ ID NO: 7, and which does not hybridize withunrelated sequences. Preferred oligonucleotide probes according to theinvention are SEQ ID NOS:12, 13, 14, 15, and 16.

In a specific embodiment, the purified polynucleotides according to thepresent invention encompass polynucleotides having at least 80% identityin their nucleic acid sequences with polynucleotide of SEQ ID NO: 7 orfragments thereof. By percentage of nucleotide identity according to thepresent invention is intended a percentage of identity between thecorresponding bases of two homologous polynucleotides, this percentageof identity being purely statistical and the differences between twohomologous polynucleotides being located at random and on the wholelength of said polynucleotides. The calculation was made according tothe software GCG and the program “gap.”

The oligonucleotide probes according to the present invention hybridizespecifically with a DNA or RNA molecule comprising all or part of thepolynucleotide of SEQ ID NO: 7 under stringent conditions. As anillustrative embodiment, the stringent hybridization conditions used inorder to specifically detect a polynucleotide according to the presentinvention are advantageously the following:

Prehybridization and hybridization are performed as follows in order toincrease the probability for heterologous hybridization:

-   -   The prehybridization and hybridization are done at 50° C. in a    -   solution containing 5×SSC and 1× Denhardt's solution.        The washings are performed as follows:    -   2×SSC at 60° C. 3 times during 20 minutes each.

The non-labeled polynucleotides or oligonucleotides of the invention canbe directly used as probes. Nevertheless, the polynucleotides oroligonucleotides are generally labeled with a radioactive element (³²P,³⁵S, ³H, ¹²⁵I) or by a non-isotopic molecule (for example, biotin,acetylaminofluorene, digoxigenin, 5-bromodesoxyuridin, fluorescein) inorder to generate probes that are useful for numerous applications.Examples of non-radioactive labeling of nucleic acid fragments aredescribed in the French Patent No. FR 78 10975 or by Urdea et al. orSanchez-Pescador et al. 1988.

Other labeling techniques can also be used, such as those described inthe French patents 2 422 956 and 2 518 755. The hybridization step maybe performed in different ways (Matthews et al. 1988). A general methodcomprises immobilizing the nucleic acid that has been extracted from thebiological sample on a substrate (nitrocellulose, nylon, polystyrene)and then incubating, in defined conditions, the target nucleic acid withthe probe. Subsequent to the hybridization step, the excess amount ofthe specific probe is discarded, and the hybrid molecules formed aredetected by an appropriate method (radioactivity, fluorescence, orenzyme activity measurement).

Advantageously, the probes according to the present invention can havestructural characteristics such that they allow signal amplification,such structural characteristics being, for example, branched DNA probesas those described by Urdea et al. in 1991 or in the European Patent No.0 225 807 (Chiron).

In another advantageous embodiment of the present invention, the probesdescribed herein can be used as “capture probes”, and are for thispurpose immobilized on a substrate in order to capture the targetnucleic acid contained in a biological sample. The captured targetnucleic acid is subsequently detected with a second probe, whichrecognizes a sequence of the target nucleic acid that is different fromthe sequence recognized by the capture probe.

The oligonucleotide fragments useful as probes or primers according tothe present invention can be prepared by cleavage of the polynucleotideof SEQ ID NO: 7 by restriction enzymes, as described in Sambrook et al.in 1989. Another appropriate preparation process of the nucleic acids ofthe invention containing at most 200 nucleotides (or 200 bp if thesemolecules are double-stranded) comprises the following steps:

-   -   Synthesizing DNA using the automated methods, such as        beta-cyanethylphosphoramidite described in 1986;    -   cloning the thus obtained nucleic acids in an appropriate        vector; and    -   purifying the nucleic acid by hybridizing to an appropriate        probe according to the present invention.

A chemical method for producing the nucleic acids according to theinvention, which have a length of more than 200 nucleotides (or 200 bpif these molecules are double-stranded), comprises the following steps:

-   -   Assembling the chemically synthesized oligonucleotides, which        can have different restriction sites at each end;    -   cloning the thus obtained nucleic acids in an appropriate        vector; and    -   purifying the nucleic acid by hybridizing to an appropriate        probe according to the present invention.

The oligonucleotide probes according to the present invention can alsobe used in a detection device comprising a matrix library of probesimmobilized on a substrate, the sequence of each probe of a given lengthbeing localized in a shift of one or several bases, one from the other,each probe of the matrix library thus being complementary to a distinctsequence of the target nucleic acid. Optionally, the substrate of thematrix can be a material able to act as an electron donor, the detectionof the matrix positions in which hybridization has occurred beingsubsequently determined by an electronic device. Such matrix librariesof probes and methods of specific detection of a target nucleic acid aredescribed in European patent application No. 0 713 016, or PCTApplication No. WO 95 33846, or also PCT Application No. WO 95 11995(Affymax Technologies), PCT Application No. WO 97 02357 (AffymetrixInc.), and also in U.S. Pat. No. 5,202,231 (Drmanac), said patents andpatent applications being herein incorporated by reference.

The present invention also pertains to a family of recombinant plasmidscontaining at least a nucleic acid according to the invention. Accordingto an advantageous embodiment, a recombinant plasmid comprises apolynucleotide of SEQ ID NO: 7 or nucleic acid fragment thereof. Morespecifically, the following plasmids are part of the invention:

-   -   DH5[alpha]-pTc45 MIT (1335 bp) and    -   Dh5[α]pTc45 MIT (239 bp).

A suitable vector for the expression in bacteria, and in particular inE. coli, is pET-28 (Novagen), which allows the production of arecombinant protein containing a 6×His affinity tag. The 6×His tag isplaced at the C-terminus or N-terminus of the recombinant polypeptide.The purified racemase is obtained by expression in E. coli transformedwith pET-28 containing the insert of the plasmid corresponding to theCNCM No. I-2344 deleted of the sequence of the signal peptide as shownon SEQ ID NO: 9 and including the six C terminal histidine residues. Theexpression of pET-28 with the insert was induced by IPTG (1 millimolar)overnight at 20° C. resulting in a soluble recombinant racemaseaccording to the invention. This racemase has a molecular weight of 45kda in 8% SDS PAGE gel (see FIG. 4 a) compared with standard molecularweight kit markers (BioLABS). After lysis of the bacterial cells by aFrench Press, followed by centrifugation (2000 g during 15 minutes), therecombinant protein was purified from the supernatant using nickel IMACchromatography. (commercialized by PHARMACIA) and eluted in 0.5 molarimidazol buffer. The yield is between 10 to 40 mg of protein for oneliter of bacterial culture at 1. OD density. The OD at the beginning ofthe induction of the expression in the recombinant bacterial wascomprised between 0.6 to 1 OD. In the culture conditions as disclosedabove, the majority of the recombinant protein produced is in a solubleform and they are not favorable for the expression of the proteins ofthe bacterial host. The estimation of the molecular weight of thepurified recombinant protein is done more or less 10% around 45 kda.

The polypeptides according to the invention can also be prepared byconventional methods of chemical synthesis, either in a homogenoussolution or in solid phase. As an illustrative embodiment of suchchemical polypeptide synthesis techniques, the homogenous solutiontechnique described by Houbenweyl in 1974 may be cited.

The polypeptides of the invention are useful for the preparation ofpolyclonal or monoclonal antibodies that recognize the polypeptides (SEQID NOS: 1, 2, 3, and 4) or fragments thereof. The monoclonal antibodiescan be prepared from hybridomas according to the technique described byKohler and Milstein in 1975. The polyclonal antibodies can be preparedby immunization of a mammal, especially a mouse or a rabbit, with apolypeptide according to the invention, which is combined with anadjuvant, and then by purifying specific antibodies contained in theserum of the immunized animal on a affinity chromatography column onwhich has previously been immobilized the polypeptide that has been usedas the antigen.

Consequently, the invention is also directed to a method for detectingspecifically the presence of a polypeptide according to the invention ina biological sample. The method comprises:

-   -   a) bringing into contact the biological sample with an antibody        according to the invention; and    -   b) detecting antigen-antibody complex formed.

Also part of the invention is a diagnostic kit for in vitro detectingthe presence of a polypeptide according to the present invention in abiological sample. The kit comprises:

-   -   a polyclonal or monoclonal antibody as described above,        optionally labeled; and    -   a reagent allowing the detection of the antigen-antibody        complexes formed, wherein the reagent carries optionally a        label, or being able to be recognized itself by a labeled        reagent, more particularly in the case when the above-mentioned        monoclonal or polyclonal antibody is not labeled by itself.

Indeed, the monoclonal or polyclonal antibodies according to the presentinvention are useful as detection means in order to identify orcharacterize a T. Cruzi strain carrying TC45 genes.

The invention also pertains to:

-   -   A purified polypeptide or a peptide fragment having at least 10        amino acids, which is recognized by antibodies directed against        a polynucleotide or peptide sequence according to the invention.    -   A polynucleotide comprising the full length coding sequence of        the Tc45 gene sequence according to the invention.    -   A monoclonal or polyclonal antibody directed against a        polypeptide or a peptide fragment encoded by the polynucleotide        sequences according to the invention.    -   A method of detecting the presence of parasite harboring the        polynucleotide sequences according to the invention in a        biological sample comprising:    -   a) contacting DNA or RNA of the biological sample with a primer        or a probe according to the invention, which hybridizes with a        nucleotide sequence;    -   b) amplifying the nucleotide sequence using said primer or said        probe; and    -   c) detecting the hybridized complex formed between said primer        or probe with the DNA or RNA.

A kit for detecting the presence of a parasite harboring thepolynucleotide sequences according to the invention in a biologicalsample, comprises:

-   -   a) a polynucleotide probe according to the invention; and    -   b) reagents necessary to perform a nucleic acid hybridization        reaction.

A method of screening active molecules for the treatment of theinfections due to a parasite, comprises the steps of:

-   -   a) bringing into contact a parasite containing the        polynucleotide sequences according to the invention with the        molecule; and    -   b) measuring an activity of the active molecule on the parasite.

An in vitro method of screening for an active molecule capable ofinhibiting a polypeptide encoded by the polynucleotide sequencesaccording to the invention, wherein the inhibiting activity of thesemolecules is tested on at least said polypeptide, comprises the stepsof:

-   -   a) providing a polypeptide according to the invention;    -   b) contacting the active molecule with said polypeptide;    -   c) testing the capacity of the active molecules, at various        concentrations, to inhibit the activity of the polypeptide; and    -   d) choosing the active molecule that provides an inhibitory        effect of at least 80% on the activity of the said polypeptide.

A test for screening the inhibiting activity of a molecule, for example,a new substrate analogue or a new antiparasitic agent, can comprise thefollowing steps:

A suitable test for testing an active molecule inhibiting thepolypeptide according to the invention is performed as follows:

-   -   The recombinant amino acid purified or native racemase is        diluted in sodium acetate buffer or Tris or phophate on Hepes        buffer at 3 micrograms per 500 microliters in the presence of 20        millmolar of beta mercepto ethanol and 10 to 80 millimolar of L        or D substrate and containing various concentrations of active        molecule to be tested. This reaction is incubated for 30 minutes        at 37° C. and stopped by heating at 80° C. Variations in optical        rotation are measured by a polarimeter.

Another embodiment of this invention provides a method for inhibitingthe activity of a parasite in vivo. The method comprises administeringto a host a parasite mitogen, which is capable of exhibiting aprotective effect, a curative effect, or preventing transmission of aparasite in the host. The parasite mitogen is administered to the hostin an amount sufficient to prevent or at least inhibit infection in vivoor to prevent or at least inhibit spread of the parasite in vivo. Theseeffects are achieved by administering the parasite mitogen to the hostin a sub-mitogenic amount, which is preferably sufficient to induce aprotective response against the parasite in the host.

The parasite mitogen employed in this invention is distinguished from an“antigen”, which is a substance that induces an immune response, such asa complete antigen that both induces an immune response and reacts withthe product of the response, or an incomplete antigen (hapten) thatcannot induce an immune response by itself, but can react with theproducts of an immune response when complexed to a complete antigen(carrier). The parasite mitogens of the present invention are thusunlike antigens, which require processing and presentation, such as (1)uptake of the antigen by antigen presenting cells (APCs); (2)internalization of the antigen in intracellular vesicles; (3)intracellular processing, which may include the unfolding of a proteinand/or partial proteolysis, with generation of immunogenic peptides; (4)binding of peptides to class II MHC molecules to form a bimolecularcomplex recognized by T cells; and (5) transport to, and display of, thecomplex on the surface of APCs. In addition, the parasite mitogensemployed in this invention do not require activation of the APCs asmanifested by the expression of: (1) adhesion molecules that promote thephysical interaction between APCs and T cells; (2) membrane boundgrowth/differentiation molecules (co-stimulators) that promote T cellactivation; or (3) soluble cytokines, such as IL-1 and TNF, as isrequired in the process for presenting antigens.

The mitogen employed in this invention is also distinguished from a“superantigen”, which is a substance that can stimulate all of the Tcells in an individual that express a particular set or family of V_(β)Tcell receptor genes. Superantigens are typically bacterial and viralproducts, and can either be soluble or cell-bound. They do not requiredegradation to peptides. Superantigens are typically presented to the Tcell receptor (TCR) on MHC molecules; however, they do not requireprocessing by antigen presenting cells (APC), as do antigens, in orderto be presented.

Thus, used herein, the term “mitogen” refers to a polyclonal activatorthat has the capacity to bind to and to trigger proliferation ordifferentiation of B lymphocytes, T lymphocytes, or mixtures thereof.Lymphocyte proliferation or transformation is the process whereby newDNA synthesis and cell division takes place in lymphocytes after astimulus of some type, resulting in a series of changes. The lymphocytesincrease in size, the cytoplasm becomes more extensive, the nucleoli arevisible in the nucleus, and the lymphocytes resemble blast cells. Theterm blast transformation is also sometimes applied to this process.Mitogens can induce proliferation in normals cells in culture.Activation of the lymphocytes thus can be characterized bytransformation of the lymphocytes into blast cells, synthesis of DNA,cell division, increased production of immunoglobulins, or increasedcytokine production. More particularly, the mitogens employed in thisinvention can stimulate whole classes of lymphocytes in this manner, andnot just clones of particular specificity. The mitogens employed in thisinvention function, therefore, in a manner similar to the effectsproduced by lipopolysaccharide (LPS) on B cells, or lectins,concanavalin A (ConA), and phytohemagglutinin (PHA) on T cells.

With these phenomena in mind, the expression “parasite mitogen”, as usedherein, means at least one protein or polypeptide found in a parasite,wherein the protein or polypeptide is capable of provoking non-specificpolyclonal activation of B lymphocytes, T lymphocytes, or mixturesthereof, in an in vitro culture of the lymphocytes in the manner similarto that just described. The protein or polypeptide comprising theparasite mitogen can be in glycosylated or non-glycosylated form. Theparasite mitogen can be in natural or recombinant form.

The term “recombinant” as used herein means that a protein orpolypeptide employed in the invention is derived from recombinant (e.g.,microbial or mammalian) expression systems. “Microbial” refers torecombinant proteins or polypeptides made in bacterial or fungal (e.g.,yeast) expression systems. As a product, “recombinant microbial” definesa protein or polypeptide produced in a microbial expression system,which is essentially free of native endogenous substances. Proteins orpolypeptides expressed in most bacterial cultures, e.g. E. coli, will befree of glycan. Proteins or polypeptides expressed in yeast may have aglycosylation pattern different from that expressed in mammalian cells.

The parasite mitogen employed in this invention can be in isolated orpurified form. The terms “isolated” or “purified”, as used in thecontext of this specification to define the purity of protein orpolypeptide compositions, means that the protein or polypeptidecomposition is substantially free of other proteins of natural orendogenous origin and contains less than about 1% by mass of proteincontaminants residual of production processes. Such compositions,however, can contain other proteins added as stabilizers, excipients, orco-therapeutics. The parasite is isolated if it is detectable as asingle protein band in a polyacrylamide gel by silver staining.

Evaluation of lymphocyte proliferation can be quantitated in an assay ofproliferative activity. For example, a radiolabelled precursor of DNA(usually tritiated thymidine) can be added to a culture medium and theamount of radioactivity incorporated into the cells subsequentlydetected. A suitable assay involves the in vitro culture of a lymphocytepopulation in the presence or absence of a mitogen for various periodsof time. The changes induced in the stimulated groups are compared withchanges in unstimulated cell populations. Radiolabelled amino acids areconvenient as they provide a means of quantitating the changes in asimple, reproducible manner. Thus, as used herein, the expression “assayof proliferative activity” means the following assay:

Assay of Proliferative Activity

In vitro proliferation is accomplished using freshly recoveredsplenocytes from BALB/c mice seeded at a density of 5×10⁴ cells/well andincubated for 24, 48 and 72 h with increasing concentrations of totalparasite supernatants or recombinant TcPa45 protein or other mitogen(0.07-200 μg/ml) with 0.5 μg/ml of the HPLC fractions, or with theconventionally used mitogens concanavalin A (10 μg/ml) andlipopolysaccharide (5 μg/ml) in 5% FCS in RPMI-1640 complete medium.T-cell depletion is accomplished by incubating freshly recovered spleencells for 30 min. at 37° C. in the presence of monoclonal antibodiesagainst Thy 1.2 and rabbit complement (Cedarlane, Le Perray en Yvelines,France). Analysis of proliferative activity of total splenocytes (5×10⁴cells/well) in the presence of 50 μg/ml enzymatically active rTcPA45 orother mitogen is also compared with the proliferation obtained using thesame amounts of rTcPA45 protein or other mitogen lacking racemaseactivity (by heating for 10 min. at 30° C. or by long term storage at 4°C.). Inhibition of proliferation is obtained by adding to the splenocytecultures 50 μg/ml rTcPA45 or other mitogen pre-incubated for 10 min. at37° C. with 1 mM inhibitor, either specific (pyrrole-2-carboxylic acid)or nonspecific (iodoacetamide or iodoacetate). Competitive assays ofproliferative activity by 50 μg/ml rTcPA45 or other mitogen are done byadding increasing concentrations of specific substrates i.e., prolineracemase substrates (L- or D-proline) for the mitogen rTcPA45 rangingfrom 3 mM to 50 mM. Controls include the incubation of splenocytes(5×10⁴ cells/well) with substrate alone, i.e., 50 mM L- or D-proline inRPMI medium alone for prline racemase. Cultures are collected after a16-hour pulse or 1 μCi/well ³H-thymidine uptake was determined in abeta-plate liquid scintillation counter (LKB-Wallac, Orsay, France). Alldata points are obtained in triplicate and the corresponding standarddeviation is calculated.

This assay of proliferative activity is used to determine whether asubstance is a parasite mitogen. This assay is also used to determine asub-mitogenic amount of the parasite mitogen. The results obtained in atypical assay of proliferative activity for two different mitogens, Aand B, are depicted in FIG. 13, which is not based on data actuallyobtained in this invention, but which is merely included forillustrative purposes to show the effects produced by mitogens afterlymphocyte activation.

Moreover, while the identification of a parasite or virus mitogen foruse in the invention is accomplished by use of the assay ofproliferative activity, it will be understood that the results obtainedwith this assay can be followed by other methods of measuring activationof lymphocytes, such as by measuring immunoglobulin production and/orcarrying out immunoglobulin specificity assays. The Elispot assaydescribed hereinafter can be used for this purpose.

As used herein, the term “sub-mitogenic amount” means an amount of theparasite mitogen, which is less than an amount of the parasite mitogenthat produces an increase in lymphocyte proliferation in the assay ofproliferative activity. Thus, the sub-mitogenic amount can be easilydetermined by carrying out the assay of proliferative activity atseveral low dosages of the parasite mitogen and noting the dosage atwhich proliferative activity first increases. The sub-mitogenic amountis an amount below the dosage at which proliferative activity firstincreases.

The sub-mitogenic amount must also be sufficient to induce protectiveimmunity against the parasite in a host to which the sub-mitogenicamount of the parasite mitogen is administered. As used herein, the term“protective immunity” refers to an adaptive (specific) immune responsecharacterized by specificity and memory in the host to which thesub-mitogenic amount of the parasite mitogen is administered. Theadaptive immune response once stimulated by an invading parasite willremember and respond more rapidly to infection so that no disease willoccur or any disease that occurs following infection will be less severeas compared to a similar infection without prior immunization accordingto the invention. Thus, the protective immunity imparted by the methodof the invention imparts protection from disease, particularlyinfectious disease, as evidenced by the absence of clinical indicationsof disease, or as evidenced by absence of, or reduction in, determinantsof pathogenicity, including the absence or reduction in persistence ofthe infectious parasite or virus in vivo, and/or the absence ofpathogenesis and clinical disease, or diminished severity thereof, ascompared to individuals not treated by the method of the invention.

The determination of a sub-mitogenic amount can readily be understood byreference to FIG. 13, which depicts two dose-response curves for twodifferent hypothetical mitogens A and B. A sub-mitogenic amount ofmitogen A would be an amount below about dose “Y” in FIG. 13, while asub-mitogenic amount of mitogen B would be an amount below dosage “X” inFIG. 13.

The TcPA45 protein of the invention is referred to as a “parasitemitogen” in a functional sense in that it is capable of activating anon-specific polyclonal response in lymphocytes in the assay ofproliferative activity. TcPA45 itself might not be a mitogen, but mayact through racemization or by binding to host molecules, which would bethe primary mitogens. Regardless, the non-specific lymphocyte activationby TcPA45 in amounts exceeding a sub-mitogenic amount would insureevasion of the parasite at the very beginning of infection.

The evasiveness and diversity of parasites has made definitive treatmentdifficult. Presented here are methods and agents for preventing thespread of parasitic and viral infections in a host, such as a human.Examples of microorganisms against which the methods and agents of theinvention are effective are the following. TABLE 2 Immune dysfunctionsobserved after mitogen-induced polyclonal activation followinginfectious processes Target Microorganism lymphocytes Acute orprogressive dysfunctions Actinomyces viscousus B ImmunosuppressionAfrican Swine Fever virus B Immunosuppression Ascaris B IgE secretin,allergy, cerebral granuloma Borrelia burgdorferi B Autoimmune arthritisCandida albicans B Granuloma formation, immunosuppression Chlamidiatrachomatis B Lymphocytosis, autoimmunity Entamoeba histolytica TImmunosuppression, disabling colitis, liver abscess Escherichia coli BToxic shock syndrome, meningitis, neurological and systemic symptomsLeishmania donovani, L major B Immunosuppression, autoimmunity Listeriamonocytogenes B and T Meningitis, immune complex formation andimmunosuppression Mycobacterium tuberculosis T Immunosuppression andautoimmune arthritis Plasmodium chabaudi, P. yoellii TImmunosuppression, autoimmunity P. falciparum B and T Immunosuppression,autoimmunity Salmonella paratyphi, S. typhimurium B Lethal scepticemia,vascular myocardial injuries, immunosuppression, autoimmunitySchistosoma mansoni, S. hematobium B Immunosuppression, megasyndromes,granuloma Staphylococcus aureaus B Toxic shock syndrome, mastitis,immunosuppression Streptococcus intermedius, S. mutans B Toxic shocksyndrome, immunosuppression S. pyogenes B and T Immunosuppression,autoimmunity Toxocara canis B Eosinophilia, lung damage, oculargranuloma, vasculitis Toxoplasma gondii B and T Encephalitis,myocarditis, immunosuppression Trypanosoma brucei B Immunosuppression,glomerulonephritis and brain lesions T. congolense B Immunosuppressin T.cruzi B and T Hypergammaglobulinemia, immunosuppression, autoimmunemyocarditis, megasyndromes

These and other substances employed as mitogens in this invention are ofparasitic or viral origin, are of natural or recombinant form, and aresimilarly capable of producing a polyclonal response in unselectedlymphocytes in the assay of proliferative activity. More particularly,while this invention has been described with reference to parasitemitogens, it is equally applicable to viral antigens. Indeed, bysubstituting “viral mitogen” for the expression “parasite mitogen” and“virus” for “parasite” in the foregoing description, the full scope ofthe subject invention will be understood. In addition, the parasitemitogens employed in this invention will exemplified with reference tothe mitogen TcPA45 protein of T. cruzi. While the parasite mitogenTcPA45 is also a racemase, it will be understood that the parasitemitogens and virus mitogens can be employed in this invention may or maynot possess racemase activity.

Following are additional examples of microorganisms against which thevaccination strategy of the invention is applicable: Infection withEpstein-Barr, influenza, herpes, and sendai viruses; non- or poorlypathogenic viruses, such as and African swine fever virus; and murineleukaemia virus. Both T-cell-dependent and independent polyclonal B-cellactivation have also been described for a variety of protozoan parasiteinfections, and these infections can also be prevented or abated by thevaccination strategy of this invention. These microorganisms includePlasmodium berghei, P. yoellii, P. chabaudi, P. falciparum and P. vivax.

In practicing the method of the invention, the parasite or viral mitogenis administered to a host using one of the modes of administrationcommonly employed for administering drugs to humans and other animals.Thus, for example, the parasite or viral mitogen can be administered tothe host by the oral route or parenterally, such as by intravenous orintramuscular injection. Other modes of administration can also beemployed, such as intrasplenic, intradermal, and mucosal routes. Forpurposes of injection, the mitogens described above can be prepared inthe form of solutions, suspensions, or emulsions in vehiclesconventionally employed for this purpose.

It will be understood that the parasite and viral mitogens can be usedin combination with other parasite or viral mitogens or otherprophylactic or therapeutic substances. For example, mixtures ofdifferent parasite mitogens or mixtures of different viral mitogens canbe employed in the method of the invention. Similarly, mixtures ofparasite and viral mitogens can be employed in the same composition. Theparasite and viral mitogens can also be combined with other vaccinatingagents for the corresponding disease, such microbial immunodominant,immunopathological and immunoprotective epitope-based vaccines orinactivated attenuated, or subunit vaccines. The parasite and viralmitogens can even be employed as adjuvants for other immunogenic orvaccinating agents.

The parasite or viral mitogen is employed in the method of the inventionin an amount sufficient to provide an adequate concentration of the drugto prevent or at least inhibit infection of the host in vivo or toprevent or at least inhibit the spread of the parasite or virus in vivo.The amount of the mitogen thus depends upon absorption, distribution,and clearance by the host. Of course, the effectiveness of the parasiteor viral mitogen is dose related. The dosage of the parasite or viralmitogen should be sufficient to produce a minimal detectable effect, butthe dosage should be less than the dose that activates a non-specificpolyclonal lymphocyte response as measured by the assay of proliferativeactivity previously described.

The dosage of the parasite or viral mitogen administered to the host canbe varied over wide limits. The parasite or viral mitogen can beadministered in the minimum quantity, which is therapeuticallyeffective, and the dosage can be increased as desired up the maximumdosage tolerated by the patient. The parasite or viral mitogen can beadministered as a relatively high sub-mitogenic amount, followed bylower maintenance dose, or the parasite or viral mitogen can beadministered in uniform dosages.

The dosage and the frequency of administration will vary with theparasite or viral mitogen employed in the method of the invention. Inthe case of the TcPA45 parasite mitogen, the sub-mitogenic amountadministered to a human can vary from about 50 ng per Kg of body weightto about 1 μg per Kg of body weight, preferably about 100 ng per Kg ofbody weight to about 500 ng per Kg of body weight. Similar dosages canbe employed for the other parasite and viral mitogens employed in thisinvention but optimum amounts can be determined with a minimum ofexperimentation using conventional dose-response analytical techniquesor by scaling up from studies based on animal models of disease.

The term “about” as used herein in describing dosage ranges means anamount that is equivalent to the numerically stated amount as indicatedby the induction of protective immunity in the host to which theparasite or viral mitogen is administered, with the absence or reductionin the host of determinants of pathogenicity, including an absence orreduction in persistence of the infectious parasite or virus in vivo,and/or the absence of pathogenesis and clinical disease, or diminishedseverity thereof, as compared to individuals not treated by the methodof the invention.

The dose of the parasite or viral mitogen is specified in relation to anadult of average size. Thus, it will be understood that the dosage canbe adjusted by 20-25% for patients with a lighter or heavier build.Similarly, the dosage for a child can be adjusted using well knowndosage calculation formulas.

The parasite or viral mitogen can be used in therapy in the form ofpills, tablets, lozenges, troches, capsules, suppositories, injectablein ingestable solutions, and the like in the treatment of cytopatic andpathological conditions in humans and susceptible non-human primates andother animals.

Appropriate pharmaceutically acceptable carriers, diluents, andadjuvants can be combined with the parasitic and viral mitogensdescribed herein in order to prepare the pharmaceutical compositions foruse in the treatment of pathological conditions in animals. Thepharmaceutical compositions of this invention contain the activemitogens together with a solid or liquid pharmaceutically acceptablenontoxic carrier. Such pharmaceutical carriers can be sterile liquids,such as water an oils, including those of petroleum, animal, vegetable,or synthetic origin. Examples of suitable liquids are peanut oil,soybean oil, mineral oil, sesame oil and the like. Water is a preferredcarrier when the pharmaceutical composition is administeredintravenously. Physiological solutions solutions can also be employed asliquid carriers, particularly for injectable solutions.

The ability of the vaccines of the invention to induce protection in ahost can be enhanced by emulsification with an adjuvant, incorporationin a liposome, coupling to a suitable carrier, or by combinations ofthese techniques. For example, the vaccines of the invention can beadministered with a conventional adjuvant, such as aluminum phosphateand aluminum hydroxide gel. Similarly, the vaccines can be bound tolipid membranes or incorporated in lipid membranes to form liposomes.The use of nonpyrogenic lipids free of nucleic acids and otherextraneous matter can be employed for this purpose.

Suitable pharmaceutical excipients include starch, glucose, lactose,sucrose, gelatine, malt, rice, flour, chalk, silica gel, magnesiumcarbonate, magnesium stearate, sodium stearate, glycerol monstearate,talc, sodium chloride, dried skim milk, glycerol, propylene glycol,water, ethanol, and the like. These compositions can take the form ofsolutions, suspensions, tablets, pills, capsules, powders,sustained-release formulations and the like. Suitable pharmaceuticalcarriers are described in “Remington's Pharmaceutical Sciences” by E. W.Martin. The pharmaceutical compositions contain an effective therapeuticamount of the parasite or viral mitogen together with a suitable amountof carrier so as to provide the form for proper administration to thehost.

The host or patient can be an animal susceptible to infection by theparasite or virus, and is preferably a mammal. More preferably, themammal is selected from the group consisting of a human, a dog, a cat, abovine, a pig, and a horse. In an especially preferred embodiment, themammal is a human.

Another aspect of the invention includes administering nucleic acidsencoding parasite and/or viral mitogens with or without carriermolecules to an individual. Those of skill in the art are cognizant ofthe concept, application, and effectiveness of nucleic acid vaccines(e.g., DNA vaccines) and nucleic acid vaccine technology as well asprotein and polypeptide based technologies. The nucleic acid basedtechnology allows the administration of nucleic acids encoding parasiteand/or viral mitogens, naked or encapsulated, directly to tissues andcells without the need for production of encoded proteins prior toadministration. The technology is based on the ability of these nucleicacids to be taken up by cells of the recipient organism and expressed toproduce a mitogen to which the recipient's immune system responds. Suchnucleic acid vaccine technology includes, but is not limited to,delivery of naked DNA and RNA and delivery of expression vectorsencoding the parasite or viral mitogen. Although the technology istermed “vaccine”, it is equally applicable to immunogenic compositionsthat do not result in a complete protective response. Suchpartial-protection-inducing compositions and methods are encompassedwithin the present invention.

Although it is within the present invention to deliver nucleic acidsencoding the parasite or viral mitogens as naked nucleic acids, thepresent invention also encompasses delivery of nucleic acids as part oflarger or more complex compositions. Included among these deliverysystems are viruses, virus-like particles, or bacteria containing thenucleic acids encoding the parasite or viral mitogen. Also, complexes ofthe invention's nucleic acids and carrier molecules with cellpermeabilizing compounds, such as liposomes, are included within thescope of the invention. Other compounds, such as molecular vectors (EP696,191, Samain et al.) and delivery systems for nucleic acid vaccinesare known to the skilled artisan and exemplified in, for example, WO 9306223 and WO 90 11092, U.S. Pat. No. 5,580,859, and U.S. Pat. No.5,589,466 (Vical patents), which are incorporated by reference herein,and can be made and used without undue or excessive experimentation.

Results indicate that intramuscular DNA vaccination protocols usingpcDNA3 vector containing the TcPA45 gene, with or without the fragmentencoding the signal peptide, are able to induce a decrease of 85% inparasitemia levels after challenge with infective forms of the parasite.Moreover, even higher levels of parasitemia control resulted whensub-mitogenic doses of the active rTcPA45 protein were injectedintraperitoneally 2 weeks before infective challenge. These obervationssupport the use of this molecule as a drug and/or immunomodulatortarget.

This invention further contemplates:

-   -   1. Any molecular modification of the gene or a fragment of the        gene encoding for a racemase/mitogen that leads to the        inhibition of the expression of the protein by the parasite or        virus (gene knock out), and further utilization of parasites or        viruses lacking those activities in vivo aiming at        immunoprotective responses.    -   2. Any molecular modification of the gene or a fragment of the        gene encoding for a racemase/mitogen that leads to the        hyperexpression of the protein by the parasite or virus (gene        transgenesis), and further utilization of the parasite or virus        to produce high amounts of the protein aiming at producing high        amounts of the native protein.    -   3. Any molecular modification of the gene or a fragment of the        gene encoding for a racemase/mitogen that leads to an        attenuation of parasite or virus infectivity, or interaction        with a host cell, and further injection of the parasites or        viruses in vivo aiming at immunoprotective responses.    -   4. Any molecular modification (for instance directed        mutagenesis) of the protein or of its active site that leads to        the inhibition of its enzymatic or its mitogenic activity and        further injection of mutated parasites or viruses in vivo aiming        at immunoprotective responses.    -   5. Use of any molecular or biochemical modification of the        enzymatic activity of the racemase (inhibition of the active        site) aiming at developing specific immunotherapy.    -   6. Any molecule or compound that inhibits the enzymatic activity        of the protein aiming at developing a drug against parasite or        virus infection or specific treatment of parasitic or viral        disease.

An example of the application of this technology to the inventionfollows:

The catalytic site of TcPA45 protein responsible for racemase activityis identified by the boxed region in FIG. 2. The catalytic sitecomprises the amino acides SPCGT. Inhibition of racemase activity, andthe consequent loss in infectivity, can be accomplished by altering thiscatalytic site in the protein or altering the corresponding nucleotidesin the gene encoding the protein. A target for alteration would be thecysteine residue. For example, changing the cysteine residue to serinedoes not significantly alter the charge on the molecule, but diminishesracemase activity. The catalytic site can be altered in other ways, suchas by the addition, deletion, or substitution of another moiety for oneof the moieties in the protein or the nucleic acid encoding the proteinso that secondary and tertiary structures are not materially altered,binding sub-units are not affected, but racemase activity is diminishedor totally lost.

Plasmids containing the polynucleotides from T. Cruzi have beendeposited at the Collection Nationale de Cultures de Microorganismes(“C.N.C.M.”) Institut Pasteur, 28, rue du Docteur Roux, 75724 ParisCedex 15, France, as follows: Plasmid Accession No. Deposit DateDH5α-pTc45MIT (239 bp) I-2221 Jun. 9, 1999 DH5α-pTc45MIT (1335 bp)I-2344 Oct. 29, 1999.

This invention will now be described with reference to the followingExamples.

EXAMPLE 1 Mice and Parasites

Trypanosoma cruzi clone CL Brener was used throughout this work.Epimastigotes were maintained by weekly passage in liver infusiontryptose medium. In vitro metacyclogenesis was performed in a proteinfree defined medium at 27° C., as previously described¹¹. Male euthymicor athymic BALB/c mice 8 weeks of age were purchased from Charles RiverLaboratories (Saint Aubin les Elbeuf, France). Male C3H/Hel mice 8 weeksof age from our animal facilities were also used.

EXAMPLE 2 Protein Fractionation

40 liters of culture supernatants from metacyclic forms, maintained foran additional 96 h at 37° C., were concentrated by vacuum dialysis anddialyzed against buffer A. HPLC was performed using a weak anionexchanger column POROS HQ-10 (Perspective Biosystems) at a flow rate of1 ml/min according to the following program: a) 10 min with buffer A; b)30 min linear gradient from buffer A to B; c) 5 min linear gradient frombuffer B to C; and d) 5 min with buffer C. One ml fractions werecollected, frozen at −80° C., lyophilized and reconstituted in H₂0 or innon-supplemented RPMI medium for in vitro proliferation assays. (Buffersused: A: 5 mM NH₄-acetate, pH 8. B: 1 M NH₄-acetate, pH 8. C: 1 M NaCl/1M NH₄-acetate, pH 8). Fractions 1 ml in volume were collected, frozen at−80° C., lyophilized and reconstituted in water or in non-supplementedRPMI medium for in vitro proliferation assays. SDS-PAGE analysis usedstandard techniques.

EXAMPLE 3 Generation of Peptides and Amino Acid Sequence Analysis

HPLC fractions 22 and 23 were pooled and fractionated by 8% SDS-PAGE.After amino black staining, the 45 kDa protein band was cut out, in-geldigested with trypsin, and submitted to reverse phase HPLC to separatepeptides. Automated Edman degradation sequence analysis was performed inthe Laboratoire de Microséquençcage de Protéines of the PasteurInstitut.

EXAMPLE 4 RNA Preparation, Reverse Transcription, PCR, and Cloning

RNA was extracted from trypomastigote forms obtained from Vero cells,using TRIzol LS reagent (Gibco) following manufacturer's instructions.Two μg from this RNA were reverse transcribed in 20 μl with SuperscriptII (Gibco) using anti-sense degenerate primer 5′TTICCRAADATIACIACGTT3′[SEQ ID NO: 12] designed from peptide 5.

PCR reaction was performed on 5 μl of cDNA using Taq polymerase (PerkinElmer) or Pfu DNA polymerase (Stratagene). The following PCR conditionsand primers were used: 1. TcPA45 gene fragment (239 bp) 30 s at 94° C.,45 s at 45° C., 30 s at 72° C. for 30 cycles followed by 10 min at 72°C.; degenerate primers: corresponding to peptide 4 (forward)5′ATHGCITTYGGIGGIAAYTTT3′ [SEQ ID NO: 13] and to peptide 5 (reverse):5′TTICCRAADATIACIACGTT3′ [SEQ ID NO: 14]. (D for A, G or T; H stands forA, C or T; M for A or C; I for inosine; R for A or G; Y for C or T). 2.

The TcPA45 coding sequence (from codons 30 to 423) was amplified using45 s at 94° C., 45 s at 50° C., 3 min at 72° C. for 20 cycles, withprimers 5′CTCTCCCATGGGGCAGGAAAAGCTTCTG3′ [SEQ ID NO: 15] and5′CTGAGCTCGACCAGATCTATCTGC3′ [SEQ ID NO: 16]. PCR products were purifiedwith Qiagen PCR extraction kit and cloned into pCR II-TOPO vector usingthe TOPO-TA cloning kit (Invitrogen) following manufacturer'sinstructions.

EXAMPLE 5 Automated Sequencing

Lambda phage and plasmid DNA were prepared using standard techniques,and direct sequencing was performed using Big Dye Terminator kit (PerkinElmer) following manufacturer's instructions. Extension products wererun for 7 h in an ABI 373B automated sequencer. Primers internal to thesequence have also been used for sequencing.

EXAMPLE 6 Genomic Library Screening

A genomic library of T. cruzi CL-Brener constructed in phage lambda FixII (from Dr. E. Rondinelli, UFRJ, Brazil) was screened using a ³²Plabelled 239 bp PCR product as a probe. Hybridization was performedusing standard conditions. Filters were scanned using a Phosporlmagerscanning unit (Molecular Dynamics). Positive phages were identified andphage DNA was prepared using standard procedures.

EXAMPLE 7 Expression Constructs and Recombinant Protein Expression

The PCR product encoding the TcPA45 gene fragment starting at codon 30was cloned in frame with a C-terminal 6× histidine tag into thepET28b(+) expression vector (Novagen). The soluble recombinant proteinwas produced in E. coli, and the soluble fraction was purified using aNi²⁺ column (Novagen) following manufacturer's instructions.

EXAMPLE 8 Racemase Activity

Demonstration of the racemase enzymatic activity of rTcPA 45 used apolarimeter. Buffer Na-acetate (pH 6), reaction Vol. 500 μl. Optimum pHwas found to be 6 and the temperature 37° C.

3 μg of rTcPA45 was diluted in 500 μl of buffer (0.2M NaOAc, 20 mMβ-mercaptoethanol, pH6.0) containing different concentrations of thesubstrate (10-80 mM or L- or D-proline). (See FIG. 4 c.) The reactionwas incubated for 30-60 minutes at 37° C. in a water bath, followed byheat inactivation of the enzyme for 10 minutes at 80° C. Each sample isdiluted to 1.5 ml with water and the mixture submitted to measurement ofoptical rotation using a polarimeter, at 365 nm. A sample without enzymeis used as a control. For optimal pH determination, buffer systems withNaOAc, phosphate, and Tris were used with pH ranging from 4.0-8.2.Combined results are shown in FIG. 4 e. Curve of temperature wasperformed between 27° C. to 47° C.

EXAMPLE 9 Mitogenic Activity

Analysis of mitogenic proliferative activity of spleen cells in vitrowith rTcPA45 (His/tag) protein.

The figure [FIG. 4] is representative of 3 experiments using differentmouse strains (C57BL/6, BLAB/c and C3H:HeJ, the latter aLPS-nonresponsive strain). Similar results were obtained with the 3strains, including C3H/HeJ, showing that our preparation is notcontaminated by bacterial LPS. The proliferation is dose-dependent andpresents a bi-modal pattern as already observed with total culturesupernatants used to purify the Tc45 protein. Best cellconcentration=5×10⁴ cells/well.

5×10⁵ naive spleen cells/well (96 well plate) were stimulated in vitrowith different doses of rTcPA45 (ranging in FIG. 4 b from 0.8 to 200μg/ml final) for 24, 48 and 72 h, at 37° C., 5% CO₂. Cultures werepulsed with ³H-Thymidine (1μ Ci/well) for 16-18 h before harvesting.³H-thymidine incorporation was obtained after counting using abeta-plate. Results present arithmetic means of c.p.m. (counts/minute)from 6 wells/dose of rTcPA45 or wells containing medium alone (+/−SD ofthe means). (FIG. 4 b)

EXAMPLE 10 Mitogenicity of rTcPA45 In Vivo Assessed by ELISPOT

BALB/c mice were injected or not with 50 μg of rTcPA45 (i.p.), andspleen cells assayed day 7 after injection. Results represent totalnumbers of spleen cells, total number of B cells producing IgM, IgG2a,or IgG2b isotypes, and total numbers of isotype-producing B cellsspecific of the protein. TABLE 1 ELISPOT assay 7 days after i.p.injection of rTc45MIT Total Number of Ig-producing B cells IgM-producingIgG2a-producing IgG2b-producing cells cells cells Non-injected 163000 ±25456 3650 ± 636  4300 ± 142 PTc45MIT 371666 ± 94495 9866 ± 3000 14633 ±3287 (50 μg/mouse) Total Number of Ig-producing B cells ANTI-rTc45MITIgM-B cells IgG2a-B IgG2b-B anti 45 cells anti 45 cells anti 45Non-injected none none none PTc45MIT 84 none none (50 μg/mouse)

It is observed 1) a 2 fold increase in total spleen cell numbers after 7days of rTcPA45 injection, 2) 3-6 fold increase in total numbers ofIg-secreting cells, and 3) less than 0.5% of IgM secreting cells aredirected to the injected protein, characterizing a mitogenic stimulationof B cells.

Mitogenicity of the rTcPA45 In Vivo (Assay)

Mice were injected or not with 50 μg of iTcPA45 (i.p.). 7 days later,spleens were removed and cell suspensions were prepared and counted.Numbers of Ig-secreting cells (total or specific to rTcPA45) weredetermined by Elispot assay, as follows:

Elispot Assay

Flat bottomed 96 well ELISA plates, were coated with either goatanti-mouse Ig or with rTcPA45 and incubated at 4° C. overnight. Plateswere blocked with PBS-gelatin, washed with PBS-Tween and with RPMImedium. 100 μl of different spleen cell concentrations per well (in RPMIcontaining 2% FCS) were incubated for 8 hours at 37° C., 5% CO₂. A rowin the plate containing serial dilutions of purified immunoglobulin(IgM, IgG2a, or IgG2b) was used as standard. After lysis of the cellswith H20-Tween, plates were then washed with PBS-Tween and incubatedwith respective biotin-labeled antibodies directed to (IgM, IgG2b, orIgG2a isotypes), overnight at 4 EC. After washing, plates were incubatedfor 45-60 minutes with avidin-alkaline phosphatase and further incubatedwith substrate (2-amino-2-methyl-propanol buffer containing BCIP) 2-4hours, 37° C. (until the spots are “dark” blue). Spots correspond toimmunoglobulin-producing B-cell (of a particular isotype) directed tothe coated antigen (here: goat anti-mouse immunoglobulins or rTcPA45).Spots are then counted and numbers corrected to total number of spleencells according to the dilution. (See Table I and FIG. 7.)

EXAMPLE 11 DNA Vaccination

8 week old BALB/c mice (5 mice/group) were injected (i.m.) once, or 3times (interval of 3 weeks) with the different constructions (100 Φlplasmid/femoral quadriceps), as follows:

-   -   a) controls: saline and rTc24    -   b) Vectors (pcDNA3 and VR1020, which is described by R. Amasamy        et al., Biochemica Biophysica Acta 1998, Vol. 1453, pp. 1-13) of        DNA vaccination containing different constructs: Long,        containing the complete sequence of rTcPA45 gene; Short,        containing the sequence of rTcPA gene without the signal        peptide, *VR1020 vector contains an additional signal peptide        (tissue Plasminogen Activating factor, TPA).    -   c) empty vectors

Mice were challenged 4 weeks after the last injection with 10⁴ infectiveforms of the parasite/mouse, and the parasitemia was scored during 35days. Serum samples were collected before challenge and assayed byWestern blot against the recombinant protein.

It is worth noting that BALB/c mice were treated by almost 2 months (9weeks) to follow the vaccination protocol and were challenged at 21 weekold. It is well known that over 10 week old mice are resistant to theexperimental infection with Trypanosoma cruzi and no mortality isobserved.

The results using this vaccination protocol revealed that 3 injectionsof pcDNA3 containing either the Short or Long constructs, or just 1injection of the same vector with the Short construct is able to reduceby more than 50% the parasitemia levels. Titres of total immunoglobulinsanti-rTcPA45, 4 weeks after the last plasmid injection:controls (salineand empty vectors):1:100; both constructs in pcDNA3 (Short and Long):1:2000, and respectively 1/1000 and :2000 for VR1020 containing the Longand Short sequences. (See FIG. 8.)

EXAMPLE 12 Southern, Western and Northern Blots

Mice were immunized intrasplenically with 10 ng protein and were boostedevery 3 weeks with 1 μg of the same preparation for 2 months to obtainpolyclonal serum containing rTcPA45-specific antibodies. Total, solubleand insoluble sonic extracts, or culture supernatants from the differentparasite forms were purified and separated by 8-10% SDS-PAGE, andproteins were electrophoretically transferred to nitrocellulosemembranes. Membranes were saturated with Tris-buffered saline and milk,incubated with polyclonal serum against rTcPA45 and developed withperoxidase-labeled secondary antibody using an ECL kit (Arnersham,Orsay, France). T. cruzi genomic DNA (10 μg) was digested withrestriction enzymes (BarntII, BglII, SalI, TaqI and PstI), separated by0.8% agarose gel electrophoresis and transferred to Hyband N+ followedby hybridization of the membrane with a ³³P-dATP-labeled probe coveringthe TcPA45-coding sequence. Total RNA was prepared from epimastigote,metacyclic and trypomastigote forms of the parasite by conventionalmethods. For northern blot analysis, 20 μg epimastigote RNA wastransferred to Hybond N+ membranes, then hybridized with single-strandedDNA complementary to the TcPA45 gene transcript, labeled withα-³²P-dCTP.

Transcript analysis through reverse transcription and PCR. Total RNA (1μg) from epimastigote, metacyclic and trypomastigote forms of theparasite were used to synthesize specific first-strand cDNA by usingoligonucleotide R300-45 (5′-TCCGTATCCATGTCGATGC-3′) [SEQ ID NO:24],located about 240 nucleotides downstream from the first ATG start codon,followed by PCR amplification using R300-45 and an oligonucleotidecorresponding to part of the T. cruzi spliced leader sequence(5′-TATTATTGATACAGTTTCTG-3′) [SEQ ID NO:25]. An internal TcPA45 fragmentof about 170 bp was then amplified using R300-45 and the oligonucleotideHI-45 (5′-CTCTCCCATGGGGCAGGAAAAGCTTCTG-3′) [SEQ ID NO:26] to demonstratethe presence of Tc45 transcript in each of the life stages analyzed.

EXAMPLE 13 Immunofluorescence

Cellular localization of TcPA45 protein in epimastigote, metacyclic andbloodstream forms of the parasite was demonstrated by indirectimmunofluorescence using polyclonal mouse serum against rTcPA45(described above) followed by 4 μg/ml Alexa 488J goat antibody againstmouse IgG (H+I), F(ab′)₂ fragment conjugate (Molecular Probes-Interchim,Montlucon, France), compared with control staining using Alexa 488™F(ab′)₂ fragment conjugate alone or after incubation of the parasiteswith chronic serum obtained from mice infected for 8 months.

EXAMPLE 14 Racemization Assays

The percent of racemization of different concentrations of L-proline,D-proline, L-hydroxy-proline and D-hydroxy-proline substrates wascalculated by incubating a 500-μl mixture of 3 μg TcPA45 and 10-80 mMsubstrate in 0.2 M sodium acetate and 25 mM β-mercaptoethanol, pH 6, for30 min at 37° C. The reaction was stopped by incubation for 10 min at80° C. Water (1 ml) was then added, and the optical rotation wasmeasured in polarimeter 241 MC (Perkin Elmer, Montignyle Bretonneux,France) at a wavelength of 365 nm, in a cell with a path length of 10cm. The percent inhibition of racemization of 80 mM L-proline wasdetermined in the presence of different concentrations of severalspecific and nonspecific inhibitors ranging in concentration from 6 mMto 100 mM. The percent racemization of 80 mM L-proline as a function ofpH was determined using 0.2 M sodium acetate, postassium phosphate andTris-HCl buffers containing 25 mM β-mercaptoethanol; reactions wereincubated for 30 min at 37° C. as described above. All reagents andinhibitors were purchased from Sigma.

Accession numbers. The GenBank accession number of T. cruzi TcPA45 isAF195522. The EMBL accession number of C. sticklandii is E10199.

EXAMPLE 15

rTcPA45 is a B cell mitogen from a pathogenic trypanosome and a noveleukaryotic proline racemase. Both mitogenic and racemase activities seemto be linked and dependent on the integrity, or the availability of theenzyme active site. Specific inhibition of the active site (usingspecific or non-specific proline racemase inhibitors), or the activesite occupancy by the substrate (competition assays using L- orD-proline), respectively, abolish or decrease B-cell mitogenicproperties of rTcPA45.

As with classical B cell mitogens, rTcPA45 mitogenicity isdose-dependent (FIG. 1). As for classical B cell mitogens, mousespecific immune responses directed to mitogenic rTcPA45 is indeedpossible (Western blot FIG. 12) following a protocol of immunizationwhich consisted of with 1 injection (i.p.) of a sub-mitogenic (10ng/mouse) dose of the protein followed by an additional boost with amitogenic dose of rTcPA45 (50 μg/mouse) one week later.

Protocol

5 groups of 6 week old Balb/c mice (3 mice per group) were immunized asfollows:

-   -   T=non immunized.    -   Vide=immunized with 50 g of empty PcDNA3 vector, i.m. per 3 week        (twice).    -   Court=immunized with 50 μg of PcDNA3-short (TcPA45 short), i.m.        per week (twice).    -   I. S.=immunized with 10 ng of rTcPA45 i.s. (first week) and 50        μg rTcPA45 i.p. (second week).    -   I.P.=immunized with 10 ng of rTcPA45 i.p. (first week) and 50 μg        rTcPA45 i.p. (second week).    -   Sera from individual mice were collected and analyzed by Western        blot:    -   80 μg of rTcPA45 were loaded onto 0.8% SDS-page gels and        transferred to Hybond membranes.

Sera were individually tested at 1/400 dilution and reactivity againstrTcPA45 revealed with anti-IgG mice immunoglobulins usingchemuluminescence.

FIG. 12 (J-14) represents individual serum reactivity beforeimmunization before challenge (J0), and after 10 days (J10) or 21 days(J21) of challenge. Challenge consisted of i.p 10000 parasites.

Additionally, rTcPA45 molecule can be considered as a target forvaccination strategies, since the previous Example using DNA-vaccinationprotocols (intramuscular injections of DNA-vectors containing the Tc45gene) was able to reduce by 85% the parasitemia levels after aninfectious challenge with the parasite. (See FIG. 14 a.)

Moreover, experiments showed that higher levels of parasitemia control(90-95%) was obtained when the submitogenic protocol of immunizationdescribed above was followed by an infectious challenge (10⁴parasites/mouse). These additional results do support the claim thatmitogenic moieties are potential targets for vaccination approachesagainst Trypanosoma cruzi infection. (See FIG. 14(b).)

In summary, we have investigated the mechanisms and consequences ofpolyclonal lymphocyte activation using the experimental model of Chagasdisease, caused by the protozoan parasite T. cruzi. As in otherinfectious processes, this disease involves extensive B- and T-cellactivation, hypergammaglobulinemia, and the establishment of chronicautoimmunity affecting the heart and the digestive tract. Trypanosomacruzi infection induces a lymphocyte blast transformation of a magnitudethat is similar to or higher than that induced by the classic polyclonalactivator LPS.

This invention involves the use of a parasite or viral mitogen as avaccinating agent without lymphocyte polyclonal activation that inhibitsa protective host specific immune response to the parasite or virus.This invention not only prevents infection by the parasite or virus, butalso avoids the negative consequences of such infection(immunosuppression, persistent infection, and susceptibility toimmunopathology and autoimmune phenomenon). This invention thusaddresses the correlation of polyclonal lymphocyte activation of theimmune system after infections with poor specific responses and severeimmunosuppression to autologous or unrelated challenges through aneffective vaccination strategy.

REFERENCES

The following publications are cited herein. The entire disclosure ofeach publication is relied upon and incorporated by reference herein.

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1-78. (canceled)
 79. A purified nucleic acid molecule comprising: (A) amolecule that hybridizes to either strand of a denatured,double-stranded DNA comprising the nucleic acid molecule of SEQ ID NO: 7under conditions of moderate stringency and that encodes an amino acidracemase activity; (B) a molecule that is derived by in vitromutagenesis from SEQ ID NO: 7 and encodes an amino acid racemaseactivity; or (C) a molecule that is degenerate from SEQ ID NO: 7 as aresult of the genetic code and that encodes an amino acid racemaseactivity.
 80. A recombinant vector that directs the expression of anucleic acid molecule of claim
 79. 81. A host cell transfected ortransduced with the vector as claimed in claim
 80. 82. A kit fordetecting a parasite, said kit comprising: (A) a polynucleotide probe,which hybridizes with the nucleic acid as claimed in claim 79 undermoderate stringency; and (B) reagents to perform a nucleic acidhybridization reaction.